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Methods Mol Biol. 2014;1086:41-52. doi: 10.1007/978-1-62703-667-2_3.

RNA structural analysis by enzymatic digestion.

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Department of Molecular Microbiology and Immunology, University of Missouri, Columbia, MO, USA.


Enzymatic probing is a rapid, straightforward method for determining which regions of a folded RNA are structurally constrained. It can be carried out using very small amounts of material, and is especially suitable for short RNAs. Here we report a protocol that we have found to be useful and readily adaptable to the evaluation of RNAs up to 150-200 nucleotides in length. Considerations for optimization are also included. In brief, the method includes folding end-labeled RNA into its native conformation, partial digestion with structure-sensitive nucleases, and identification of the cleavage sites by electrophoretic separation of the cleavage fragments.

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